1. Field of the Invention
The present invention relates to a method for assay of an enzyme and an apparatus therefor. More specifically, the present invention relates to a method for assay of an enzyme in which an enzyme on a solid body surface is assayed by measuring a pH change of a substrate solution that occurs depending on the activity of the enzyme on a solid body surface; and to an apparatus suitably used for the method. The method for assay of an enzyme of the present invention is mainly used in the field of clinical laboratory examinations, but it is also applicable to such fields as experimental medicine, veterinary medicine, biochemistry, and pharmacology.
2. Discussion of the Related Art
In recent years, Helicobacter pylori (hereinafter referred to as "H. pylori") has drawn attention as a bacterium involved in the onset of gastric ulcer and duodenal ulcer. H. pylori, which is found in the mucosal epithelia of the stomach and the duodenum, is reported to decompose urea in the stomach and the duodenum by a large amount of urease it expresses on its membrane or secretes extracellularly. This produces a high concentration of ammonia and creates a neutral environment suitable for the growth of H. pylori, which injures epithelial cells of the organs [Kohashi, Rinsho Shokaki Naika, 8, 489 (1993)]. Therefore, H. pylori testing has increasingly become important for diagnosis of gastric and duodenal ulcers.
There are several practical methods for testing H. pylori in the stomach and the duodenum (hereinafter referred to as "the gastrointestinal tract"), including (1) culturing, (2) tissue staining, (3) the rapid urease test, (4) endoscopic method using dyes, and (5) the antibody test [Ishii, Rinsho Shokaki Naika, 8, 503 (1993)].
Of the existing methods (1) to (5) above, the culturing method (1) is to detect H. pylori by isolation culture, etc. using a mucosal tissue specimen. Although this method can give the most reliable results, it injures patient's organ because a mucosal tissue specimen should be obtained. The culturing method also requires skilled operation and takes several days. The tissue staining method (2) also requires collection of a mucosal tissue specimen and therefore injures patient's organ. It also requires skilled operation and takes about one day. In the rapid urease test (3), a mucosal tissue specimen is collected and placed in a medium containing urea and an indicator (e.g., phenol red), and a pH changes of the medium caused by urease secreted by H. pylori are visualized by an indicator [Yamamoto et al., Rinsho Shokaki Naika, 8, 517 (1993)]. Although results can be obtained within several minutes to hours with simple operation, this method, like methods (1) and (2), needs to collect mucosal specimen and injures the organs. The endoscopic method using a dye (4) is to visually observe the distribution of H. pylori in the gastrointestinal tract with an endoscopy after spreading the urea solution containing an indicator, such as toluidine blue or phenol red, in the gastrointestinal tract [Kori et al., Rinsho Shokaki Naika, 8, 525 (1993)]. Although this method is advantageous in that H. pylori distribution in the gastrointestinal tract can be observed without injuring patient's organ, it is necessary to adjust the gastrointestinal pH near the color change range in advance by, for example, administering an H.sub.2 blocker, because the color change range of an indicator is limited, and false-positive or false-negative responses may occur if the pH is not adjusted appropriately. The antibody test (5) is to determine anti-H. pylori antibodies in serum by enzyme immunoassay [Takagi, Rinsho Shokaki Naika, 8, 511 (1993)]. Because this method measures antibodies against H. pylori, rather than H. pylori itself, it assesses past history of H. pylori infection, and is incapable of determining whether or not H. pylori is present in the patient body.
As stated above, there are various methods for testing H. pylori, each having both advantages and disadvantages. From the clinical viewpoint, required is a method that can detect H. pylori in situ at a targeted site of the gastrointestinal tract at the time of endoscopy without sampling tissue specimens. Such direct in situ detection method is also desired for detecting other microorganisms in various organs, because at present bacteriological approaches, which comprise specimen sampling, in vitro culturing and microscopic observation, are used in most cases. Further, there is also a great practical need for direct measurement of an enzyme at a targeted site of an organ or solid culture medium, because collection of specimens should be unavoidable in most existing assays of enzymes.